Neuro-immune interactions inchemical-induced airway hyperreactivity

Fien C. Devos1, Brett Boonen2, Yeranddy A. Alpizar2, Tania Maes3, Valérie Hox4,Sven Seys4, Lore Pollaris1, Adrian Liston5, Benoit Nemery1, Karel Talavera2,Peter H.M. Hoet1 and Jeroen A.J. Vanoirbeek1

Affiliations: 1Centre for Environment and Health, Dept of Public Health and Primary Care, KU Leuven,Leuven, Belgium. 2Laboratory for Ion Channel Research and TRP Research Platform (TRPLe), Dept of Cellularand Molecular Medicine, KU Leuven, Leuven, Belgium. 3Laboratory of Pneumology, Dept of RespiratoryMedicine, Ghent University, Ghent, Belgium. 4Laboratory of Clinical Immunology, Dept of Microbiology andImmunology, KU Leuven, Leuven, Belgium. 5Laboratory of Genetics of Autoimmunity, Dept of Microbiology andImmunology, KU Leuven, Leuven, Belgium.

Correspondence: Jeroen A.J. Vanoirbeek, KU Leuven, Department of Public Health and Primary Care, Centrefor Environment and Health, Herestraat 49 Mailbox 706, 3000 Leuven, Belgium.E-mail: jeroen.vanoirbeek@kuleuven.be

ABSTRACT Asthma may be induced by chemical sensitisers, via mechanisms that are still poorlyunderstood. This type of asthma is characterised by airway hyperreactivity (AHR) and little airwayinflammation. Since potent chemical sensitisers, such as toluene-2,4-diisocyanate (TDI), are also sensoryirritants, it is suggested that chemical-induced asthma relies on neuro-immune mechanisms.

We investigated the involvement of transient receptor potential channels (TRP) A1 and V1, majorchemosensors in the airways, and mast cells, known for their ability to communicate with sensory nerves,in chemical-induced AHR.

In vitro intracellular calcium imaging and patch-clamp recordings in TRPA1- and TRPV1-expressingChinese hamster ovarian cells showed that TDI activates murine TRPA1, but not TRPV1. Using an in vivomodel, in which an airway challenge with TDI induces AHR in TDI-sensitised C57Bl/6 mice, wedemonstrated that AHR does not develop, despite successful sensitisation, in Trpa1 and Trpv1 knockoutmice, and wild-type mice pretreated with a TRPA1 blocker or a substance P receptor antagonist. TDI-induced AHR was also abolished in mast cell deficient KitWsh/Wsh mice, and in wild-type mice pretreatedwith the mast cell stabiliser ketotifen, without changes in immunological parameters.

These data demonstrate that TRPA1, TRPV1 and mast cells play an indispensable role in thedevelopment of TDI-elicited AHR.

@ERSpublicationsChemical-induced AHR relies on neuro-immune interactions, involving lymphocytes, TRP channelsand mast cells http://ow.ly/Z4LtH

Copyright ©ERS 2016

This article has supplementary material available from erj.ersjournals.com

Received: Oct 28 2015 | Accepted after revision: March 02 2016 | First published online: April 28 2016

Support statement: The project was supported by a grant of the Interuniversity Attraction Poles programme/BelgianState/Belgian Science Policy project P7/30 and by grants from the research council of KU Leuven (GOA/14/011, EF/95/010 and PF-TRPLe) and the Flemish Research Foundation (G.A022.11, G.0896.12 and G.0C77.15). B. Boonen is fundedby a PhD grant from the Agency for Innovation by Science and Technology. Y.A. Alpizar holds a postdoctoral mandatefrom KU Leuven. Funding information for this article has been deposited with FundRef.

Conflict of interest: None declared.

380 Eur Respir J 2016; 48: 380–392 | DOI: 10.1183/13993003.01778-2015

ORIGINAL ARTICLEASTHMA

http://crossmark.crossref.org/dialog/?doi=10.1183/13993003.01778-2015&domain=pdf&date_stamp=2016-04-28mailto:jeroen.vanoirbeek@kuleuven.behttp://ow.ly/Z4LtHhttp://ow.ly/Z4LtHerj.ersjournals.comhttp://www.crossref.org/fundref/

IntroductionAsthma is a chronic airway disease [1] which can be distinguished into various (overlapping) categories,depending on aetiology or patterns of airway inflammation [2, 3].

Most experimental asthma research has focused on atopic asthma, such as that caused in adults or children byIgE-mediated allergy to biological agents, most often using models in which mice or other animals are sensitisedto ovalbumin (OVA) or other proteins [4, 5]. However, up to half of people with asthma are not atopic [6] andthe aetiology and pathophysiology of this type of asthma remain largely unelucidated. In this regard, onepossible avenue of research is that of asthma caused by exposure to reactive chemicals [7], as may occur in theworkplace. Such chemical-induced occupational asthma has similar clinical features to atopic asthma, but theimmune and nonimmune processes leading to this form of asthma have been less well investigated [8].

In addition, asthma can present with varying types and degrees of airway inflammation, as evidenced bythe cellular composition of sputum [2]. Atopic asthma is traditionally characterised by eosinophilinflammation, but other patterns exist as well. One of these subtypes is paucigranulocytic asthma, whichdiffers from classic atopic asthma by a low degree of airway inflammation. According to SIMPSON et al. [9],in 30% of asthma cases airway hyperreactivity (AHR) can exist in the absence of inflammatory cells in theairways. The mechanisms of this form of asthma are not well understood.

We undertook the present mechanistic study to improve our understanding of the pathophysiology of aclinically important fraction of asthma, namely asthma that is not dependent on IgE responses and notcharacterised by much cellular inflammation in the airways. For this we have used a well-validated mousemodel of chemical-induced immune-mediated asthma based on sensitisation to toluene-2,4-diisocyanate(TDI), a well-described occupational asthmogen [8, 10–12].

Diisocyanates, including TDI, have a long history of use for a large number of industrial applications, andthey represent a leading cause of chemical-induced occupational asthma [13, 14]. A recent longitudinalstudy of workers exposed to low airborne levels of TDI at a modern polyurethane foam factory in EasternEurope has shown that over the first year of follow-up 14% of workers developed respiratory symptoms,TDI-specific IgGs and/or changes in spirometry, all indicative of early TDI-related health effects.Moreover, workers that were lost to follow-up (25%) were more likely to have had asthma symptoms [15].Another recent study, evaluating the long-term outcome of diisocyanate-induced asthma, has shown that50% of diisocyanate asthmatics remain symptomatic, with worsening of lung function and need ofcontinuous anti-asthmatic therapy after an exposure-free period of 12 years, thus indicating the long-termmorbidity of occupational asthma caused by diisocyanates [16].

Several researchers have sought to discover individual risk factors and mechanisms leading to TDI-inducedasthma in humans. An important observation has been that the risk of TDI-induced asthma wasassociated with a polymorphism of the neurokinin 2 receptor (NK2R), the receptor for neuropeptides thatare released from activated airway sensory neurons, thus suggesting a neurogenic mechanism inTDI-induced asthma. This NK2R 7853G>A polymorphism was associated with higher serum vascularendothelial growth factor levels, and thus airway inflammation in TDI-exposed workers [17]. In healthyhuman subjects, activation of transient receptor potential channels (TRP)A1 or TRPV1, via inhalation ofirritant agonists (cinnamaldehyde or capsaicin), has been shown to evoke cough, a reflexive airwayresponse, which may also contribute to asthmatic symptoms [18]. Capsaicin treatment has been shown toreduce nasal hyperreactivity in patients with idiopathic rhinitis, via ablation of the TRPV1-substance Pnociceptive signalling pathway in the nasal mucosa, providing further evidence for an involvement of TRPand neurogenic inflammation in human respiratory diseases [19].

Besides being a potent sensitiser, TDI is an extremely potent sensory irritant. Exposure to TDI leads tostimulation of trigeminal nerve endings present in respiratory and nasal mucosa [20, 21], thereby inducingthe local release of tachykinin neuropeptides, including substance P, neurokinin A and calcitonin-gene-relatedpeptide [22, 23]. This probably results from stimulation of TRP channels [24].

Since TDI is both a potent immune sensitiser and sensory irritant, this chemical agent provides a uniqueopportunity to investigate the interaction between immunological and neurological processes. It has indeedbeen proposed that asthma is an immune-neuronal disorder [25], like other inflammatory conditions [26].However, the link between activation of neuronal receptors and immune responses is not well establishedin asthma.

Combining our mouse model of chemical-induced asthma with in vivo and in vitro studies of TRPA1 andTRPV1 activation [27–29], we tested the hypothesis that stimulation of sensory TRP channels criticallymodulates chemical-induced airway responses. We also investigated the role of mast cells, because mastcells are located in the vicinity of sensory nerve fibres in the airways [25] and they can thus immediatelyrespond to released neuropeptides.

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Here, we first demonstrate that TDI activates TRPA1, but not TRV1 in vitro, and then that in our in vivomodel, activation of TRPA1 (direct) and TRPV1 (indirect), together with activation of mast cells,contributes to the development of AHR. We conclude that neuro-immune interactions, involving TRPA1,TRPV1 and mast cells, are crucial in developing nonatopic irritant-induced AHR, even in the absence ofcellular manifestations of inflammation.

Materials and methodsA complete description of the materials and methods is available in the online supplementary material.

In vitro experimentsTDI-induced activation of TRPA1 or TRPV1 was investigated by intracellular calcium (Ca2+) imaging andpatch-clamp experiments on Chinese hamster ovarian (CHO) cells selectively expressing murine (m)TRPA1 or mTRPV1. A tetracycline-regulated system was used for inducible expression of TRPA1 orTRPV1 in CHO cells. TRPA1 currents were recorded in the cell-attached patch-clamp configuration.

Experimental protocols of mouse experimentsAll experimental procedures performed in mice were approved by the KU Leuven local ethics committeefor animal experiments (P166-2012).

Our model of chemical-induced asthma, as described previously in Balb/c mice [11], was applied toC57Bl/6 wild-type mice. Briefly, mice were sensitised to TDI by applying 20 µL TDI (1% in acetone/oliveoil (AOO)) to the dorsum of both ears on days 1 and 8; nonsensitised controls received similarapplications of the vehicle only. On day 15, mice were challenged by means of an intranasal instillation of30 µL of 0.1% TDI or vehicle (AOO). 1 day later, the main physiological outcome, i.e. nonspecific airwayreactivity to methacholine, was measured using the flexiVent (Scireq, Montreal, QC, Canada) [30], and thefollowing immunological readouts were obtained: cell counts and distribution in bronchoalveolar lavage(BAL) fluid, total serum IgE, lymphocyte subpopulations in retro-auricular lymph nodes and their ex vivocytokine release.

We compared the responses obtained in wild-type mice to those obtained in knockout mice devoid ofTRPA1 (Trpa1-/-), TRPV1 (Trpv1-/-), mast cells (KitWsh/Wsh) or lymphocytes (Rag2-/-). We also testedresponses in wild-type mice having received pharmacological agents before the challenge with TDI:HC030031 (TRPA1 antagonist), RP67580 (substance P receptor antagonist) or ketotifen (mast cellstabiliser); or during the challenge: allyl isothiocyanate (TRPA1 agonist) or capsaicin (TRPV1 agonist).

Statistical analysisElectrophysiological measurement data were analysed using the WinASCD software package (KU Leuven,Leuven, Belgium). Dose–response curves (AHR) were analysed using two-way parametric ANOVA, followedby a Bonferroni multiple comparison post hoc test. All other data were analysed using one-way parametricANOVA, followed by a Bonferroni multiple comparison post hoc test.

ResultsTDI activates TRPA1 in vitroIn vitro Ca2+ imaging experiments showed that 0.001% TDI caused a significant increase in intracellularCa2+ concentration in mTRPA1-expressing CHO cells, but not in mTRPV1-expressing cells (onlinesupplementary fig. S1). 0.001% toluene diamine (TDA), which is the hydrolysis product of TDI,containing two amine groups instead of reactive isocyanate groups, was ineffective on both mTRPA1- andmTRPV1-expressing cells (online supplementary fig. S1).

Furthermore, cell-attached patch-clamp recordings in mTRPA1-expressing CHO cells showed that theapplication of 0.001% TDI leads to enhanced channel activation (fig. 1a-c) and an increase in the meancurrent (fig. 1e). The presence of functional TRPA1 was confirmed by applying 100 μM menthol togetherwith TDI, which causes a “flickering” of the current (fig. 1a, d) that is characteristic of the blocking effectof menthol on TRPA1 [27]. Taken together, these data demonstrate that TDI directly activates TRPA1, butnot TRPV1.

TDI causes AHR only after prior sensitisation to TDIIntranasal instillation of 0.1% TDI induced AHR to methacholine 24 h later, only if mice had beenpreviously sensitised to TDI (TDI/TDI) (fig. 2a; for individual responses see online supplementary fig. S2).TDI-sensitised and TDI-challenged mice showed increased levels of total serum IgE (fig. 2b), increasednumbers of CD4+ T-helper (Th)-cells, CD8+ cytotoxic T-cells and B-cells (CD19+) in the auricular lymphnodes draining the site of sensitisation (fig. 2c), along with increased releases of both Th2 (interleukin

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(IL)-13 and IL-10) and Th1 (interferon (IFN)-γ) cytokines upon incubation of these lymphocytes withconcanavalin A in vitro (fig. 2d). Thus, these mice showed immunological changes, confirming that theyhad been immunologically sensitised [31].

Of note, the AHR in TDI-sensitised and TDI-challenged mice was not accompanied by increases ininflammatory cells (neutrophils, eosinophils or lymphocytes) in BAL fluid (fig. 2e). In other words, TDIinduced sensitisation-dependent AHR without signs of cellular inflammation in the airways in C57Bl/6 mice.

Involvement of TRPA1 and TRPV1 in TDI-induced AHRTo test whether TRPA1 or TRPV1 has a role in generating AHR, we compared the methacholineresponses of Trpa1 and Trpv1 knockout mice with those of similarly and concurrently treated wild-type

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mice. In contrast to wild-type mice, both TDI-sensitised and TDI-challenged Trpa1 knockout and Trpv1knockout mice did not develop AHR (fig. 3a and fig. 4a; for individual responses see onlinesupplementary fig. S2). This lack of an AHR response in both Trpa1 and Trpv1 knockout mice was notdue to absence of sensitisation, because their immune responses did not differ from those of similarlytreated wild-type mice (fig. 3b–d and fig. 4b–d).

To confirm the role of TRPA1, we assessed the effect of pharmacological inhibition of the TRPA1receptor. As seen in Trpa1 knockout mice, TDI-sensitised wild-type mice pretreated with a selectiveTRPA1 blocker (HC030031) 30 min prior to the TDI challenge, did not develop AHR (fig. 5a; forindividual responses see online supplementary fig. S2), although immune-sensitisation was not affected(online supplementary fig. S3a and b).

Since activation of TRPA1 and TRPV1 is known to induce the release of the neuropeptide substance P[32], we sought to determine whether substance P is involved in the observed TDI-induced respiratoryresponses. TDI-sensitised wild-type mice, having received an intraperitoneal injection of RP67580, anantagonist of the neurokinin 1 receptor (NK1R; the receptor for substance P), 30 min prior to the TDIchallenge, did not develop AHR 24 h later (fig. 5b; for individual responses see online supplementary fig.S2). Again, this could not be attributed to the NK1R antagonist having abolished immune-sensitisation(online supplementary fig. S3c–e).

Thus, contrary to control wild-type mice, mice deficient in TRPA1 or TRPV1 or wild-type mice in whichthe TRPA1 channel or the receptor responding to neuropeptides released by sensory neurons had beenblocked, did not develop AHR in response to a TDI challenge, even though they had been successfullysensitised to TDI.

These results provide evidence for a crucial role of TRPA1 and TRPV1 activation in TDI-induced AHR.Nevertheless, to exclude that the mere activation of these TRP channels leads to AHR in TDI-sensitised

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FIGURE 2 Toluene-2,4-diisocyanate (TDI) causes airway hyperresponsiveness after prior sensitisation. a) Airway resistance (Raw) in response tomethacholine; b) total serum IgE levels; c) number of T-helper cells (CD4+), cytotoxic T-cells (CD8+) and B-lymphocytes (CD19+); cytokine levels ofd) interleukin (IL)-13, e) interferon (IFN)-γ and f) IL-10; g) differential cell count in the bronchoalveolar lavage (BAL) fluid. Data are presented asmean±SD, n=6–9 per group. AOO: acetone/olive oil; WT: wild-type. **: p

subjects, we substituted the intranasal challenge with TDI with an intranasal instillation of allyl isothiocyanate(AITC), a potent agonist of TRPA1 and capsaicin, a potent agonist of TRPV1. In TDI-sensitised wild-typemice, AHR was absent 24 h after instillation of AITC or capsaicin (fig. 6a and b; for individual responses seeonline supplementary fig. S2), despite evidence of successful sensitisation (online supplementary fig. S4),although ex vivo cytokine release was not increased in TDI/AITC and TDI/capsaicin mice (fig. 6d). Thus, it isnot sufficient to activate TRPA1 or TRPV1 to induce AHR in TDI-sensitised mice.

Conversely, to test if the development of AHR necessitates an antigen-specific challenge, we gaveTDI-sensitised mice a first challenge with AITC (nonspecific antigen) followed 1 week later by a secondchallenge either with TDI (specific antigen) or again with AITC. AHR was only induced after the challengewith TDI, i.e. in the TDI/AITC/TDI mice (fig. 6c; for individual responses see online supplementaryfig. S2). All TDI-sensitised mice showed humoral (IgE) and cellular (T- and B- lymphocytes) evidence ofimmune sensitisation (online supplementary fig. S4), although the increased ex vivo release of IL-13, IFN-γand IL-10 occurred in TDI/AITC/TDI mice and not in TDI/AITC/AITC mice, suggesting that the cytokineproduction depends on a challenge with the specific sensitiser (fig. 6d).

Involvement of immune cells in TDI-induced AHRTaken together, the above results allowed us to conclude, first, that TRPA and TRPV1 are necessary, butnot sufficient, to induce AHR in TDI-sensitised animals and, second, that a specific adaptive immuneresponse is involved and necessary to induce AHR in such TDI-sensitised animals. Previous research onour mouse model has shown that lymphocytes are crucially involved in the development of TDI-induced

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FIGURE 3 Involvement of transient receptor potential A1 (TRPA1) channels in toluene-2,4-diisocyanate (TDI)-induced airway hyperresponsiveness:Trpa1 knockout (KO) mice. a) Airway resistance (Raw) in response to methacholine in Trpa1 KO mice; b) total serum IgE levels; c) number ofT-helper cells (CD4+), cytotoxic T-cells (CD8+) and B-lymphocytes (CD19+); cytokine release of d) interleukin (IL)-13, e) interferon (IFN)-γ andf) IL-10. Data are presented as mean±SD, n=5–8 per group. WT: wild-type; AOO: acetone/olive oil. *: p

asthma in Balb/c mice [33, 34]. To determine whether this is also the case in the current model ofchemical-induced AHR, using C57Bl/6 mice, we used Rag2 knockout mice, lacking mature lymphocytes.Indeed, AHR was absent 24 h after instillation of TDI in TDI-sensitised Rag2 knockout mice (onlinesupplementary fig. S5a; for individual responses see online supplementary fig. S2). As expected, IgE levelswere not increased in these mice (online supplementary fig. S5b).

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FIGURE 4 Involvement of transient receptor potential V1 (TRPV1) channels in toluene-2,4-diisocyanate (TDI)-induced airway hyperresponsiveness:Trpv1 knockout (KO) mice. a) Airway resistance (Raw) in response to methacholine in Trpv1 KO mice; b) total serum IgE levels; c) number of T-helpercells (CD4+), cytotoxic T-cells (CD8+) and B-lymphocytes (CD19+); cytokine release of d) interleukin (IL)-13, e) interferon (IFN)-γ and f) IL-10. Data arepresented as mean±SD, n=5–6 per group. WT: wild-type; AOO: acetone/olive oil. **: p

Since neurons have been proposed to directly communicate with mast cells [25], we also assessed the roleof the latter in our model of chemical-induced AHR, by testing mast cell deficient mice (KitWsh/Wsh mice).In contrast to wild-type mice, no induction of AHR was observed in TDI-sensitised and TDI-challengedKitWsh/Wsh mice, even though they were responsive to methacholine (fig. 7a; for individual responses seeonline supplementary fig. S2). TDI-sensitised and TDI-challenged mast cell knockout mice showed thesame humoral, cellular and ex vivo changes indicative of sensitisation as similarly treated wild-type mice(fig. 7b–d). To further validate the involvement of mast cells in TDI-induced AHR, wild-type mice weretreated with the mast cell stabiliser ketotifen 30 min prior to the TDI challenge. In these TDI-sensitisedmice, mast cell stabilisation abolished the induction of AHR 24 h after the TDI challenge (fig. 8; forindividual responses see online supplementary fig. S2), while immune sensitisation was unaffected (onlinesupplementary fig. S6). These results demonstrate that mast cells are critical for the development of AHRwhen TDI-sensitised mice are challenged with TDI.

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FIGURE 6 Induction of airway hyperresponsiveness depends on an allergen-specific challenge. Airwayresistance (Raw) in response to methacholine in wild-type (WT) mice after a first challenge with a) allylisothiocyanate (AITC) or b) capsaicin (CAP) and c) after a second challenge with either acetone/olive oil (AOO),AITC or toluene-2,4-diisocyanate (TDI); cytokine release of d) interleukin (IL)-13, e) interferon (IFN)-γ andf) IL-10. Data are presented as mean±SD, n=5–8 per group. **: p

DiscussionTo explore the interplay between neural and immunological mechanisms in asthma, we investigated thecritical determinants of the respiratory responses to TDI, a chemical irritant and sensitiser that is knownto cause immune-mediated asthma. We used a well-validated experimental model [11, 33].

The novelty of our work lies in the demonstration that TDI can directly activate mTRPA1, but notmTRPV1, and that, interestingly, both TRPA1 and TRPV1 are critical (although not sufficient) for the

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FIGURE 7 Involvement of mast cells in toluene-2,4-diisocyanate (TDI)-induced airway hyperresponsiveness:KitWsh/Wsh mice. a) Airway resistance (Raw) in response to methacholine in KitWsh/Wsh mice; b) total serum IgElevels; c) number of T-helper cells (CD4+), cytotoxic T-cells (CD8+) and B-lymphocytes (CD19+); cytokinerelease of d) interleukin (IL)-13, e) interferon (IFN)-γ and f) IL-10. Data are presented as mean±SD, n=5–10per group. WT: wild type; KO: knockout; AOO: acetone/olive oil. **: p

induction of AHR in our model. We also established convincingly that mast cells are crucially involved inthe induction of this AHR.

In our model of chemical-induced asthma, TDI-sensitised mice develop AHR 24 h after an intranasal TDIchallenge. This response of the lower airways is indicative of the occurrence of a nasobronchial cross-talk,which is probably mediated by sensory nerves that are abundantly expressed in the upper airways. Theexistence of such cross-talk was demonstrated by HENS et al. [35] in an allergic OVA model in animals inwhich asthmatic responses were produced in spite of interruption of the continuity between the nose andlungs by means of a tracheostomy. The AHR that developed rapidly in these tracheostomisedOVA-sensitised mice after a nasal challenge with OVA was attributed to pulmonary upregulation ofsubstance P and activation of the NK1 receptor; hence neurogenic lung inflammation originating in thenose [35]. Thus, both the findings of HENS et al., who used a model of atopic asthma, and our presentobservations in a model of chemical-induced asthma, support the concept of the “united airways”, from thenose to the lungs. Furthermore, our study provides novel mechanistic insight by demonstrating that thecross-talk operates through activation of TRPA1 and TRPV1 channels and with involvement of mast cells.

Using in vitro intracellular Ca2+ imaging, we showed channel activity in mTRPA1-expressing cells, but notin mTRPV1-expressing cells, in response to TDI. Direct evidence for the implication of TRPA1 was thenobtained using electrophysiology studies, which were not performed for TRPV1 due to itsunresponsiveness to TDI in the Ca2+ imaging experiments. Importantly, a previous study suggested thatTDI activates TRPA1 [36], but this contention was based on Ca2+ imaging experiments, which provideonly indirect indication of channel activity. Here, we used patch-clamp recordings and demonstrated, forthe first time, that TDI activates TRPA1 channels and that this activation needs the presence of theisocyanate moieties, since TDA (having amine groups) was inactive.

Complementary to the in vitro studies, in vivo respiratory physiology experiments were performed, usingthe flexiVent to assess changes in nonspecific airway reactivity to methacholine, 1 day after the intranasalinstillation [30]. The principal breakthrough that emerges from our studies is that both TRPA1 andTRPV1 are essential for the development of AHR after a challenge with TDI in TDI-sensitised mice. Ourfindings in Trpa1 and Trpv1 knockout mice and the validation using pharmacological agents, a TRPA1antagonist or a NK1R antagonist, convincingly prove the involvement of TRPA1, TRPV1 and theneuropeptide substance P in immune-mediated chemical-induced AHR. Importantly, our experimentswith TRP agonists (AITC and capsaicin) given to TDI-sensitised mice demonstrate that induction of AHRonly occurred in a context of specific immune sensitisation, since AHR was only induced if the animalswere challenged with the agent to which they had been sensitised. An involvement of TRPA1 inallergen-induced leukocyte infiltration and AHR has been reported, albeit without much mechanisticexplanation, in a murine OVA model [37]. Moreover, in contrast with the latter study, we have used amodel without cellular influx in the airways. Therefore, we were able to simulate paucigranulocytic asthma,a relevant condition that has hitherto hardly been studied. Furthermore, our model allowed us todisentangle the airway reactivity response from the immune-mediated inflammatory changes.

In addition, TRPV1 has been proposed to play a role in the development of allergic asthma [38, 39],although this was disputed by some others [37, 40]. In the current study, we show that TRPV1 is notactivated directly by TDI, but is essential for the induction of AHR. However, the importance of TRPV1activation can be attributed to its immunomodulatory function. Recently, TRPV1 has been demonstratedto regulate the activation and inflammatory properties of CD4+ T-cells [41]. Furthermore, it has been

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FIGURE 8 Involvement of mast cells in toluene-2,4-diisocyanate (TDI)- induced airway hyperresponsiveness:pharmacological blockade. Airway resistance (Raw) in response to methacholine in wild-type (WT) mice treatedwith either ketotifen (Keto) or vehicle (Veh). n=5–6 per group. AOO: acetone/olive oil **: p

reported that IL-13, a key cytokine in AHR development, increases the level of TRPV1 in the murine lungand can induce increased TRPV1 expression in the bronchial epithelium of mice, linking TRPV1 to IL-13induced features of asthma. Indeed, inhibition of TRPV1 has shown to attenuate IL-13 induced AHR,airway inflammation, goblet cell metaplasia and subepithelial fibrosis [38]. As such, we hypothesise thatIL-13, released by lymphocytes or mast cells, can stimulate TRPV1. Previously we have described theimportance of CD4+ T-cells and the Th2-cytokine IL-13 in our model of chemical-induced asthma, whichcould thus explain why AHR is abolished in TRPV1-deficient mice [33].

Furthermore, it has been shown that inflammatory mediators, such as prostaglandins, leukotrienes andbradykinin released by mast cells can reduce the activation threshold of TRPV1 [42, 43], and thatneuropeptides released from sensory nerves can sensitise TRPV1 and enhance afferent excitability [44]. Theinflammatory conditions can thus contribute to the activation and involvement of TRPV1 in our model.

In our search for the mechanism of enhanced nonspecific airway reactivity shortly after an antigen-specificchallenge we also focused on mast cells, because of their prominent role in asthma [45] and because of thepostulated interplay between mast cells and sensory neurons [46]. Our findings with mast cell deficientmice (KitWsh/Wsh mice), validated by experiments using a mast cell stabiliser, demonstrate that functionalmast cells are essential for the development of chemical-induced AHR after a TDI challenge inTDI-sensitised mice. We have previously reported the importance of mast cells in a model of nonallergicAHR [29], and this has also been shown in mouse models of atopic asthma [47, 48]. In the case of atopicasthma, activation of mast cells by allergen cross-linking of the IgE-bound high-affinity IgE receptor(FcɛRI) leads to the release of various pro-inflammatory mediators that increase the sensitivity of airwaysmooth muscle to nonspecific stimuli, such as methacholine [45]. How mast cells become activated andwhich mediators they release in our model remain to be investigated. Based on previous evidence [49], arole for allergen cross-linking with IgEs seems unlikely. However, there is no doubt that the adaptiveimmune system is also involved, both because the induction of AHR relies on an antigen-specificchallenge and because our model is dependent on properly functioning lymphocytes, as shown here withRag2 knockout mice and previously in severe combined immunodeficiency disease mice [11] and inadoptive lymphocyte transfer experiments [33, 34].

What we do know is that mast cells are located in the vicinity of nerve fibres present in the airways [50],and this close association with sensory nerves allows mast cells to respond immediately to neuropeptidesreleased from nearby sensory nerve endings. SCHEERENS et al. [22] suggested that tachykinin neuropeptides,such as substance P, were involved in TDI-induced tracheal hyperreactivity as measured ex vivo, and theyproposed, without giving evidence, that neurogenic inflammation resulted from the release of bioactivemediators by mast cells. Human and rodent mast cells do express neuropeptide receptors, including thesubstance P receptor NK1, and high concentrations of substance P induce mast cell degranulation [51]. Incontrast, low concentrations of substance P were shown to prime mast cells by lowering the threshold forsubsequent stimulation [52]. Conversely, mast cell activation and subsequent mediator release can sensitiseafferent sensory fibres, by lowering their stimulation threshold [46]. In summary, various lines of evidencesuggest the existence of bidirectional interaction pathways between mast cells and airway sensory nerves.

In conclusion, our results demonstrate that the induction of airway hyperreactivity by a chemical sensitisersuch as TDI relies both on a specific adaptive immune response (that operates via lymphocyte activation)and on neuro-immune interactions, with crucial involvement of TRPA1, TRPV1 and mast cells. Ourfinding that TDI-induced asthma relies on neuro-immune mechanisms, involving both TRPA1, TRPV1and mast cells, may lead to novel personalised strategies for treating patients with paucigranulocyticasthma. Patients with this phenotype of asthma may benefit from therapies targeting mast cell–TRPinteractions, via the use of neuropeptide receptor blockers, mast cell stabilisers or TRPA1 blockers.

AcknowledgementsThe authors thank Rudi Vennekens (Laboratory for Ion Research and TRP Research Platform, Dept of Cellular andMolecular Medicine, KU Leuven, Leuven, Belgium) for providing the Trpa1 and Trpv1 knockout mice.

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Neuro-immune interactions in chemical-induced airway hyperreactivityAbstractIntroductionMaterials and methodsIn vitro experimentsExperimental protocols of mouse experimentsStatistical analysis

ResultsTDI activates TRPA1 in vitroTDI causes AHR only after prior sensitisation to TDIInvolvement of TRPA1 and TRPV1 in TDI-induced AHRInvolvement of immune cells in TDI-induced AHR

DiscussionReferences